斑馬魚 myosin heavy chain cDNA 之選殖及其表現之研究

Abstract

斑馬魚受精卵發育到約10小時就可以觀察到體節（somite）的形成，而somite是由paraxial mesoderm發育而成，在隨後的胚胎發育過程，somite 將分化成為肌肉、骨骼及皮膚等組織。肌凝蛋白 (myosin）是形成肌肉所需的結構蛋白質，其重要功能之一為參與橫紋肌的收縮。目前所發現的肌凝蛋白共有14種，以MyosinⅡ為例，是由二個相同的myosin heavy chain及四個不同的myosin light chain次單元所組成。 本論文由孵化後一個月大斑馬魚所抽得的Poly(A)+ mRNA 所備製之λ gt10 cDNA library，篩選到一 2,045 bp 大的cDNA clone。經核酸及轉譯的氨基酸序列之分析及比對，此2Kb大小的cDNA片段和自鯉魚(Cyprinus carpio)選殖的myosin heavy chain最為相似，此二者核酸序列之相同性為 76.2%，氨基酸序列相同性為 83.5%，相似性則為88％。利用北方式轉印法（Northern blotting）來研究 myosin heavy chain mRNA 在斑馬魚不同胚胎發育時期之表現得知，有一 6.5Kb大的 mRNA 在孵化前後最為顯著的表現，此 mRNA 之量卻隨發育以至於成魚確有減少的趨勢。由原位雜合反應（whole mount in situ hybridization）實驗得知，此myosin heavy chain mRNA表現在 pharyngula 時期胚胎之體節（somite）及孵化後仔稚魚之骨骼肌上。另外此mRNA亦表現在孵化後四天大的仔稚魚之胸鰭肌肉上。同時原位雜合反應染色的結果顯示此myosin heavy chain mRNA主要是表現在fast muscle fiber 之部位，其是孵化前後胚胎移動力的主要來源。

The first somite appears at 10 hr post-fertilization during zebrafish embryonic development. Somites, derived from paraxial mesoderm, differentiate into muscle,cartilage and dermis in the course of development.Myosin,one of the main muscular structural proteins, primarily functions as the source of contraction force for muscle movement.Approximately 14 classes of myosin molecules have been identified from diverse organisms. For example, myosin Ⅱis consisted of two identical heavy chains and four different light chain subunits. A 2 kb cDNA was cloned from a λgt10 cDNA library, which was obtained from poly(A)+ mRNA in one-month old zebrafish larvae.Nucleic acid and deduced amino acid sequence analyses showed that the 2 kb cDNA shared high similarity with that of fast skeletal muscle myosin heavy chain from carp (Cyprinus carpio).They shared 76% nucleic acid sequence identity,83.5% amino acid sequence identity, and 88% amino acid sequence similarity.The expression of myosin heavy chain mRNA during zebrafish development was analyzed by Northern blotting. Expression of a 6.5 kb mRNA was maximal at the time of hatching and then decreased as the development proceeds, from larval to adult stage.Whole mount in situ hybridization showed that myosin heavy chain mRNA were localized at somite region of pharyngula stage embryos and skeletal muscle region of hatching larvae, at the pectoral fins of 4-days old hatching larvae. Furthermore, results from whole mount in situ hybridization also revealed that myosin heavy chain mRNA were mainly expressed in the fast muscle fibers, which are believed to be the primary production sites of contraction force of embryos at hatching.